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Cyanobacterial Lactate Oxidases Serve as Essential Partners in N … · Cyanobacterial Lactate...
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Cyanobacterial Lactate Oxidases Serve as Essential Partnersin N2 Fixation and Evolved into Photorespiratory GlycolateOxidases in Plants W
Claudia Hackenberg,a Ramona Kern,a Jan Hüge,b Lucas J. Stal,c Yoshinori Tsuji,d Joachim Kopka,b
Yoshihiro Shiraiwa,d Hermann Bauwe,a and Martin Hagemanna,1
a University of Rostock, Plant Physiology Department, 18051 Rostock, GermanybMax-Planck-Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germanyc Department of Marine Microbiology, Netherlands Institute of Ecology (NIOO-KNAW), Centre for Estuarine and Marine Ecology,
4400 AC Yerseke, The Netherlandsd Laboratory of Plant Physiology and Metabolism, Graduate School of Life and Environmental Sciences, University of Tsukuba,
Tsukuba 305-8572, Japan
Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and
green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N2-fixing
cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The
GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low
GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis
thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity
ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a
cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally
in nitrate-containing medium but died under N2-fixing conditions. Cultivation under low oxygen rescued this lethal
phenotype, indicating that N2 fixation was more sensitive to O2 in the Dlox Nostoc mutant than in the wild type. We propose
that LOX primarily serves as an O2-scavenging enzyme to protect nitrogenase in extant N2-fixing cyanobacteria, whereas in
plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.
INTRODUCTION
Cyanobacteria evolved approximately three billion years ago and
were the first organisms that performed oxygenic photosynthe-
sis. This important metabolic process was later transferred into a
eukaryotic host through an endosymbiotic event, eventually lead-
ing to the evolution of algae and land plants (Mereschkowsky,
1905; Margulis, 1970; Reyes-Prieto et al., 2007). Hence, many
plant genes originated from the cyanobacterial endosymbiont,
including those coding for proteins involved in photosynthesis
and many other metabolic and regulatory functions (Martin
et al., 2002). Phylogenetic studies have indicated that the primary
endosymbiont was closely related to extant filamentous, het-
erocystous, N2-fixing cyanobacteria (Deusch et al., 2008), such
as Nostoc (Anabaena) sp strain PCC 7120 (hereafter Nostoc).
All organisms performing oxygenic photosynthesis use the
Calvin-Benson cycle for CO2 fixation with ribulose-1,5-bis-phos-
phate carboxylase/oxygenase as the carboxylating enzyme,
producing two molecules of 3-phosphoglycerate. In addition to
its carboxylase activity, ribulose-1,5-bis-phosphate carboxylase/
oxygenase acts as an oxygenase and produces 2-phospho-
glycolate (2PG) in the presence of O2. 2PG is toxic and be-
comes converted to 3-phosphoglycerate by a process known
as photorespiration (Husic et al., 1987; Bauwe et al., 2010).
Mutations in the enzymatic steps of the photorespiratory cycle in
most cases result in lethal phenotypes at ambient CO2 levels but
can be rescued under high CO2 levels, when the oxygenase
reaction is suppressed (Blackwell et al., 1988; Somerville, 2001).
This high-CO2-requiring (HCR) phenotype is characteristic of
mutants defective in 2PG metabolism and is known as the
photorespiratory phenotype. Accordingly, in the current atmos-
phere, plants can perform oxygenic photosynthesis only in the
presence of a fully operational photorespiratory cycle. This is true
for both C3 plants and C4 plants. Mutants of the C4 plant maize
(Zea mays) with deletion of the photorespiratory enzyme glyco-
late oxidase (GOX) also exhibit an HCR phenotype (Zelitch et al.,
2009).
Because cyanobacteria possess an efficient inorganic carbon
concentrating mechanism (CCM), it was assumed that they
do not perform photorespiratory 2PG metabolism (reviewed in
Colman, 1989; Giordano et al., 2005). Cyanobacterial CCM
mutants exhibit an HCR phenotype (reviewed in Kaplan and
Reinhold, 1999; Badger et al., 2006). More recent studies have
1Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Martin Hagemann([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.088070
The Plant Cell, Vol. 23: 2978–2990, August 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.
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revealed that active photorespiratory 2PGmetabolism is present
in the cyanobacterium Synechocystis sp strain PCC 6803 (here-
after Synechocystis). This organism employs three routes for
2PG detoxification: (1) a plant-like 2PG cycle, (2) the glycerate
pathway, and (3) the decarboxylation of glyoxylate via formate.
The combined loss of all three routes resulted in an HCR
phenotype and demonstrated the essential function of photo-
respiratory 2PG metabolism in Synechocystis, despite the pres-
ence of a CCM. These findings led to the hypothesis that primary
endosymbiosis conveyed not only oxygenic photosynthesis but
also an ancient photorespiratory 2PG metabolism into the eu-
karyotic host (Eisenhut et al., 2008a).
Several differences between the photorespiratory 2PG me-
tabolism of Synechocystis and land plants have been noted
(reviewed in Bauwe et al., 2010). For example, the oxidation of
glycolate to glyoxylate in Synechocystis (and many other cya-
nobacteria) is mediated by two glycolate dehydrogenases
(GlcDs) that use NAD(P)+ as cofactor. Similarly, in green algae,
this reaction is also performed byGlcD (Codd et al., 1969; Nelson
and Tolbert, 1970; Frederick et al., 1973). Correspondingly, the
knockout of GlcDs in Synechocystis (Eisenhut et al., 2008a) or in
the green alga Chlamydomonas reinhardtii (Nakamura et al.,
2005) results in an HCR phenotype. In land plants, peroxisome-
localized GOX catalyzes the glycolate oxidation, usingmolecular
oxygen as a cosubstrate. Five isoforms of GOX proteins are
imported into the peroxisome of Arabidopsis thaliana. Among
them, isoform 2, GOX2 (At3g14415), is thought to play a major
role in the photorespiratory glycolate oxidation step (Reumann
et al., 2004; Bauwe et al., 2010). The diversity of photorespiratory
strategies among oxygenic phototrophic organisms could reflect
a stepwise loss, modification, and/or acquisition of enzymes.
In a recent phylogenetic study of enzymes possibly involved in
the plant-like 2PG metabolism (Kern et al., 2011), we found that
the genomes of N2-fixing cyanobacteria and green algae contain
genes similar to both types of glycolate-oxidizing enzymes, GlcD
and plant-like GOX. We hypothesized that the plant GOX family
could possibly originate from an ancestral cyanobacterial pro-
tein. Here, we report on a functional characterization of the
corresponding GOX-like proteins from the N2-fixing Nostoc
strain (No-LOX, gene all0170 or lox) and C. reinhardtii (Cr-LOX).
Additionally, we analyzed the importance of the No-LOX for the
diazotrophic growth of Nostoc. Our results suggest that an
ancestral cyanobacterial LOX may have evolved to perform two
different functions. In extant N2-fixing cyanobacteria such as
Nostoc, it serves as an O2-scavenging enzyme, protecting nitro-
genase. After endosymbiotic transfer, the enzyme was succes-
sivelymodified in the plant lineage to use glycolate as its preferred
substrate in the photorespiratory cycle.
RESULTS
Phylogenetic Analysis of GOX
To analyze the evolutionary history of GOX enzymes in silico, we
extended an earlier study (Kern et al., 2011) using Arabidopsis
GOX2 (At3G14415) as a target sequence in BLASTP searches to
identify a broad range of related proteins in bacteria, including
cyanobacteria, algae, and plants. These putative GOX enzymes
included theNostoc protein encoded by gene all0170,whichwas
used as a target sequence in a second set of BLASTP searches.
An unrooted phylogenetic tree (Figure 1), constructed from the
Figure 1. Phylogenetic Relationship between GOX and LOX Proteins.
Neighbor-joining tree of putative GOX or LOX proteins [(S)-2-hydroxy-acid oxidase, EC 1.1.3.15] based on 23 sequences. Numbers at the node indicate
bootstrap values (percentage) for 1000 replicates. The distance scale (substitutions per site) is shown in the top left-hand corner. Asterisks refer to
biochemically characterized enzymes (Macheroux et al., 1992; Maeda-Yorita et al., 1995; Eprintsev et al., 2009). Proteins shown in boldface were
analyzed in this study. The alignment used for this analysis is available as Supplemental Data Set 1 online.
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identified sequences, all belonging to a protein family of (S)-2-
hydroxy-acid oxidases (EC 1.1.3.15), displayed two main clus-
ters. One cluster includes the structurally and biochemically
characterized GOX from spinach (Spinacia oleracea; Lindqvist,
1989) and many other plants together with uncharacterized
cyanobacterial and algal proteins, which may be considered as
GOX-like sequences. The second cluster included many bacte-
rial proteins known to exhibit LOX activity, for example, Aero-
coccus viridans LOX (Maeda-Yorita et al., 1995). Their close
relationship is also reflected by the similar structures of their
active sites. Selected bacterial, cyanobacterial, algal, and plant
proteins from the two clusters possess only a few alterationswith
respect to the amino acid residues in their active sites (Figure 2).
The existence of a clade containing only cyanobacterial pro-
teins and proteins from photosynthetic eukaryotes (green plants,
green algae, red algae, and brown algae), which is statistically
supported by bootstrap values of 100% (Figure 1), is indicative of
a cyanobacterial origin of all these proteins in eukaryotic photo-
trophs. Correspondingly, the cyanobacterial protein fromNostoc
(All0170) has a higher overall similarity to the Arabidopsis GOX2
and the spinach GOX (70 and 62% sequence identity, respec-
tively) compared with the LOX enzymes from the heterotrophic
bacteria Lactococcus lactis and A. viridans (57 and 55% se-
quence identity, respectively) (see Supplemental Figure 1 online).
Interestingly, genes encoding cyanobacterial GOX-like pro-
teins were exclusively found in the genomes of N2-fixing spe-
cies; they were absent from the genomes of nondiazotrophic
cyanobacteria. It should be noted that, in addition to these
putative GOX-encoding sequences, all genomes of N2-fixing
cyanobacteria also harbor one or two genes coding for glyco-
late dehydrogenases that are highly similar to the photorespi-
ratory GlcD1 and GlcD2 proteins from Synechocystis (Eisenhut
et al., 2008a). The coexistence of genes coding for GlcDs (e.g.,
alr5269 - glcD1 and all4443 - glcD2 in Nostoc) and GOX-like
proteins (e.g., all0170 in Nostoc) in all sequenced genomes of
N2-fixing cyanobacteria was unexpected and raised two ques-
tions. Are cyanobacterial GOX-like proteins capable of oxidiz-
ing glycolate to glyoxylate? What are the cellular functions of
these proteins?
Biochemical Analyses of GOX-Like Proteins in Vitro
To verify the enzymatic activity of the cyanobacterial GOX-like
proteins in comparison to a plant GOX, the proteins from Nostoc
and GOX2 from Arabidopsis were overexpressed in Escherichia
coli using the corresponding gene (Nostoc) or cDNA (Arabidop-
sis) in combination with the overexpression vector pET-28a.
Virtually pure His-tagged proteins were obtained (see Supple-
mental Figure 2 online) and examined for catalytic activities with
a range of potential substrates, each at 5 mM concentration
(Table 1). Both enzymes catalyzed oxidase reactions, consuming
O2 and producing H2O2 as byproduct, but neither showed any
dehydrogenase activity with NAD+ or NADP+. In this experiment,
the cyanobacterial enzyme showed an;200-fold higher activitywith L-lactate in comparison to glycolate, whereas the plant At-
GOX2 was ;10-fold more active with glycolate than L-lactate.The clear preference for L-lactate over glycolate showed that
the cyanobacterial enzyme is a LOX rather than aGOX; therefore,
it was named No-LOX. Neither of the two enzymes accepted
D-lactate as a substrate (Table 1), which has been reported
previously for plant and algal GOX (Frederick et al., 1973).
Figure 2. Comparative Analysis of Amino Acid Exchanges in the Active Sites of GOXs and LOXs.
The given amino acid positions refer to the enzyme from Nostoc (No-LOX, All0170; the full alignment is shown Supplemental Figure 1 online). The
residues involved in the binding of FMN are highly conserved between both enzyme types (Lindqvist and Brändén, 1989; Maeda-Yorita et al., 1995;
Stenberg et al., 1995; Stenberg and Lindqvist, 1997; Leiros et al., 2006). Amino acids presumably involved in substrate binding are marked in gray.
Arrows mark amino acid changes in No-LOX variants by site-specific mutagenesis.
Table 1. Enzyme Activities of Recombinant No-LOX and At-GOX2 with
Different Substrates
Substrate (5 mM)
Relative Specific Activity (%)
No-LOX At-GOX2
Glycolate 0.44 6 0.06 100
L-lactate 100 12.58 6 5.29
D-lactate 0 0
Glyoxylate 15.33 6 3.92 0
Glycerate 5.89 6 2.77 9.96 6 5.61
Hydroxypyruvate 0.33 6 0.11 18.81 6 4.22
Enzyme activities were measured under the standard assay conditions
described in Methods. One hundred percent activity is defined for 5 mM
of the substrates L-lactate (No-LOX: 14.73 6 3.59 mmol min�1 mg�1) orglycolate (At-GOX2: 24.27 6 5.00 mmol min�1 mg�1). Parameters repre-sent the mean 6 SD of at least three independent enzyme preparations.
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No-LOX, but not At-GOX2, showed clear oxidase activity with gly-
oxylate. To a minor degree, both enzymes also accepted glycer-
ate and hydroxypyruvate as substrates (Table 1).
Next, we examined the kinetic parameters of these two en-
zymes in addition to those of the homologous protein from the
green alga C. reinhardtii. GOX-like proteins from green algae
could shed light on where in the phylogenetic tree enzyme
activity evolution occurred (Figure 1). Sequence analyses of
several independent cDNA clones, obtained from total RNA ofC.
reinhardtii grown under photoautotrophic, low-carbon condi-
tions, revealed that the nucleotide sequence differs from that in
the database (XP_001703481) by six additional nucleotides. This
is probably due to the misannotation of a splice site in the
corresponding gene (accession number of the corrected cDNA
sequence at DDBJ: AB610509). As found before with the
cyanobacterial enzyme No-LOX, the enzyme from Chlamydo-
monas has higher affinities for L-lactate than for glycolate as
substrate (Table 2) and was named Cr-LOX. With respect to the
calculated substrate-saturated rates, the estimated kinetic pa-
rameters of the enzymes confirmed the results obtained from the
experiment with fixed substrate concentrations (Table 1). The
Vmax of L-lactate oxidation by No-LOX was >200-fold higher than
that of glycolate oxidation (Table 2). By contrast, At-GOX2
exhibited a 40 times higher Vmax for glycolate than for L-lactate.
The affinity of No-LOX for L-lactate but also for glycolate is
significantly higher than that of At-GOX2 (Table 2). With respect
to all four kinetic parameters, the enzyme from Chlamydomonas
is positioned between No-LOX and At-GOX2, but with the ex-
ception of the Km for glycolate it is more similar to the cyano-
bacterial enzyme.
Additionally, the affinity of No-LOX for the cosubstrate O2 was
estimated. No-LOX activity was followed under decreasing
oxygen concentrations at saturating L-lactate concentrations.
These experiments revealed a very high affinity for O2 (Km value
of 0.296 0.14mMO2; n=21) of the recombinant No-LOXprotein.To identify the amino acid exchanges that were most impor-
tant to L-lactate versus glycolate preference, we compared the
likely substrate binding site of No-LOXwith those of the bacterial
LOX and plant GOX enzymes and found alterations in four
positions, three of which corresponded to amino acids involved
in flavin mononucleotide (FMN) and/or substrate binding (posi-
tions 82, 112, and 212 in Figure 2). Next, we replaced these three
amino acids in the No-LOX sequence, both individually and in
combinations of two or all three, with the corresponding amino
acids in the plant GOX consensus sequence. The resulting seven
mutated No-LOX variants were overexpressed in E. coli, purified,
and examined for changes in their reactivities with glycolate and
L-lactate (Table 3). With the exception of the variant M82T, which
showed higher activities with both substrates in comparison to
wild-type No-LOX, the oxidation of L-lactate was considerably
impaired in all variants. By contrast, glycolate reactivities im-
proved in most variants, including the triple mutant. The double
mutants M82T/L112W and L112W/F212V showed up to 10-fold
increased GOX activities. Moreover, the LOX/GOX activity ratios
of all variants decreased considerably and came very close to
that of At-GOX2 in the double mutant L112W/F212V and the
triple mutant (Table 3).
Complementation of the Double Mutant DglcD1/D2 of
Synechocystis by No-LOX
Previously, we have shown that deletion of GlcD in Synechocys-
tis, a cyanobacterium not capable of N2 fixation and missing a
Table 2. Kinetic Parameters of Recombinant No-LOX, Cr-LOX, and At-GOX2
L-Lactate Glycolate
Enzyme Km (mM) Vmax (mmol min�1 mg�1) Km (mM) Vmax (mmol min�1 mg�1)
No-LOX 0.039 6 0.007 12.73 6 1.55 0.233 6 0.05 0.049 6 0.021
Cr-LOX 0.081 6 0.027* 10.59 6 0.46 1.244 6 0.063* 0.189 6 0.059*
At-GOX2 0.356 6 0.182* 0.742 6 0.036* 1.906 6 0.64* 35.64 6 11.16*
Kinetic parameters were calculated by nonlinear regression fit to the Michaelis-Menten equation (Sigma Plot software) or by linear regression analysis
of the double-reciprocal data pairs (Lineweaver-Burk). Parameters represent the mean 6 SD of at least three independent enzyme preparations.
Statistically significant differences from No-LOX (asterisk) are indicated (P < 0.1 or better).
Table 3. Enzyme Activities of Wild-Type Enzymes and Amino Acid
Substitution Variants of No-LOX with Glycolate and L-Lactate as
Substrates
Activity (mmol min�1 mg�1) Ratio
Enzyme Variant
Glycolate
(5 mM)
L-Lactate
(5 mM)
L-Lactate/
Glycolate
Wild-type enzymes
No-LOX 0.06 6 0.01 14.73 6 3.59 245.5
Cr-LOX 0.16 6 0.06 9.75 6 0.46 60.9
At-GOX2 24.27 6 5.00 3.05 6 1.28 0.1
Single substitution in No-LOX
M82T 0.55 6 0.10 23.34 6 2.76 42.5
L112W 0.06 6 0.02 7.69 6 0.48 120.2
F212V 0.16 6 0.03 1.43 6 0.30 9.0
Double substitution in No-LOX
M82T, L112W 0.11 6 0.06 0.47 6 0.04 4.2
M82T, F212V 0.47 6 0.13 7.57 6 1.10 16.3
L112W, F212V 0.60 6 0.13 0.38 6 0.07 0.6
Triple substitution in No-LOX
M82T, L112W, F212V 0.26 6 0.04 0.11 6 0.03 0.4
Enzyme activities were measured using the standard assay conditions
described in Methods. The activities of the unchanged recombinant
enzymes from Nostoc, Arabidopsis, and Chlamydomonas (wild-type
enzymes, Table 1) are shown for comparison. Parameters represent the
mean 6 SD of at least three independent enzyme preparations.
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GOX-like protein, resulted in an HCR phenotype (Eisenhut et al.,
2008a). The Synechocystis double mutant DglcD1/D2 was cho-
sen as a host for complementation with No-LOX, in comparison
with At-GOX2 as a control, to evaluate the potential involvement
of No-LOX in photorespiratory glycolate oxidation in vivo. The
genotypes of strains expressing the genes all0170 (No-LOX) or
the cDNA for At3g14415 (At-GOX2) were characterized by PCR
(Figure 3A). These analyses verified the complete segregation of
the glcD1 and glcD2mutations, as observed in the parental strain
DglcD1/D2, and the presence of the No-LOX expression vector
(in the complemented strain DglcD1/D2+No-LOX) or the At-GOX2
expression vector (in the complemented strain DglcD1/D2+At-
GOX2). We also confirmed that neither of the overexpression
vectors was present in the parental double mutant. Expression
levelswere examinedby immunoblotting using specific antibodies
raised against No-LOX and At-GOX2 (Figure 3B). Cross-reacting
signals were obtained only in the complemented strains, not in the
wild type or the cells of the noncomplemented parental double
mutant. The observed expression levels of No-LOX or At-GOX2,
respectively, were unaffected by cultivation at low or high CO2conditions (LC or HC).
Previous results showed that stepwise increases in glycolate
accumulation in the single and double glcD-mutants of Syn-
echocystis (DglcD2 < DglcD1 < DglcD1/D2) were accompanied
by stepwise decreases in growth rates, ranging from the slightly
impaired growth of the single mutants to the HCR phenotype
of the DglcD1/D2 double mutant (Eisenhut et al., 2008a). The
expression of At-GOX2 or No-LOX complemented this HCR
phenotype (Figure 4). The No-LOX–expressing double mutant
was able to grow (m = 0.006 6 0.002 h21) in a similar manner tothe DglcD1 single-mutant (m = 0.005 6 0.002 h21), suggestingthat the glycolate-to-glyoxylate conversion rate of No-LOX
is similar to that of Synechocystis GlcD2 in vivo. Indeed, the
internal glycolate concentration in the No-LOX–expressing strain
decreased by ;60%, compared with the DglcD1/D2 doublemutant (Figure 4C), which was similar to the glycolate levels
reported for the single mutant DglcD1 (Eisenhut et al., 2008a). In
correlation with the much higher activity of At-GOX2 observed
with glycolate, the strain expressing this plant enzyme showed
significantly higher growth rates, reaching those of wild-type
cells (m = 0.011 6 0.002 h21). Notably, this strain accumulatedonly traces of glycolate, similar to the Synechocystis wild type
(Figure 4).
Generation and Characterization of a Nostoc DloxMutant
To analyze the function of No-LOX in its natural host, Nostoc, a
targeted mutation of all0170 (lox) was performed. We obtained
several independent spectinomycin (Sp)-resistant and Suc-
resistant clones. A genotypic characterization of these lines by
PCR (see Supplemental Figure 3 online) revealed that the wild-
type copy was completely absent in the Nostoc Dlox mutant
(Dall0170::Sp). Only disruptedmutant gene copieswere detected.
The phylogenetically related GOX proteins play an important
Figure 3. Genotypic Characterization of Strains Used in the Complementation Experiment of the Synechocystis DglcD1/D2 Double Mutant with No-
LOX or At-GOX2.
(A) Genotypic characterization of DglcD1/D2+No-LOX and DglcD1/D2+At-GOX2. Complete segregation of the genes glcD1 and glcD2 in the parental
Synechocystis strain DglcD1/D2 and the complemented strains, respectively, and detection of all0170 (encoding No-LOX) and At3g14415 (encoding At-
GOX2) in the complemented strains was verified with gene-specific primers (see Supplemental Table 2 online). M, size marker (l-DNA EcoRI/HindIII);
WT, wild type.
(B) Immunoblotting analysis with protein extracts from cells of Synechocystis wild type, DglcD1/D2, DglcD1/D2+No-LOX, and DglcD1/D2+At-GOX2 to
confirm the expression of No-LOX and At-GOX2 in the respective complemented strains with the corresponding antibodies. Cells were cultivated at
either CO2-enriched (HC) or ambient air (LC) conditions. Ten micrograms of total soluble protein fraction was applied per lane. The heterologously
expressed enzymes served as positive controls (His-No-LOX and His-At-GOX2).
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role during the photorespiration of land plants, and its deletion
results in an HCR phenotype (Zelitch et al., 2009). Therefore, we
first examined whether the Nostoc mutant Dlox expresses an
HCR phenotype similar to that of the GlcD-deficient mutants
of Synechocystis. After cultivation in nitrate-containing BG11
medium under both LC and HC conditions, however, the Dlox-
mutant exhibited growth rates similar to wild-type Nostoc.
Due to the restriction of GOX-like proteins to extant cyano-
bacteria capable of fixing atmospheric N2 (Figure 1), we rea-
soned that these enzymes may be most important under
diazotrophic growth conditions. For example, No-LOX could
play a role in the metabolism of the Nostoc heterocysts. These
are specialized cells that harbor nitrogenase and are respon-
sible for the fixation of N2 (reviewed in Kumar et al., 2010). Since
nitrogenase is extremely sensitive to O2, heterocysts provide
the anoxic environment required for this enzyme to function. As
an O2-consuming enzyme, No-LOX could possibly contribute
to a decrease in O2 partial pressure inside heterocysts. To test
this hypothesis, we transferred cells from the Nostoc wild type
and mutant Dlox to diazotrophic growth conditions in liquid and
on solid media (Figure 5). During the first 48 h following the shift
to nitrate-free conditions, the mutant showed similar growth
rates compared with the wild type (Figure 5A). After 72 h, while
wild-type cells continued to grow, the growth rate of the Dlox
mutant decreased, and the cells bleached completely after
120 h. The same phenomenon was observed when the mutant
Dlox was grown on agar plates: Dlox formed yellowish colonies
in the first week but completely bleached out after 2 weeks
in the absence of a combined nitrogen source, while wild-type
colonies were capable of growing (Figure 5B). These results
show that the Nostoc Dlox mutant was unable to grow diazo-
trophically.
To assess the failure of theNostoc Dloxmutant in fixing N2, we
quantified the heterocyst formation. Light microscopy analyses
48 h after the shift to nitrate-free conditions revealed that
filaments of the mutant Dlox contained morphologically normal
heterocysts in the same frequency as the wild-type Nostoc
(9.45%6 0.49% heterocysts in filaments of Dlox, 9.3%6 0.24%heterocysts in wild-type filaments).
Next, we shifted wild-type Nostoc and Dlox cells to nitrate-free,
microaerobic conditions (maintained by flushing bicarbonate-
supplemented cultures continuously with N2) to verify that the
inability of Dlox to grow diazotrophically was caused by the
inability of this mutant to protect nitrogenase from O2 inhibition.
Under these conditions, the mutant did not bleach and grew at
levels similar to thewild type, indicating that they had switched to
diazotrophic growth (Figure 5C) and indicating a possible role of
No-LOX in the protection of nitrogenase by the scavenging of O2in heterocysts.
To obtain additional evidence to test this hypothesis, we
performed acetylene reduction assays to measure nitrogenase
activity in vivo. After 24 and 72 h in nitrate-free medium, BG110,
aliquots of cells were taken and incubated at different oxygen
concentrations during nitrogenase activity measurements. De-
spite the relatively high deviations of the nitrogenase activities,
the two data sets showed similar changes in the oxygen sensi-
tivity of nitrogenase in intact filaments. Nostoc wild-type cells
exhibited maximum nitrogenase activities at ambient conditions
of 20%O2 (up to 160mmolC2H4 g21 chlorophyll a h21), and lower
rates were observed at both lower and higher O2 concentrations
(Table 4). By contrast, the nitrogenase activity of the Dloxmutant
was highest in the absence of O2. With increased O2 concentra-
tions, the nitrogenase activity of Dlox decreased. In 20% O2 of
ambient air, the nitrogenase activity of the mutant Dlox was
significantly lower than in the Nostoc wild-type cultures.
Figure 4. Phenotypic Characterization of Synechocystis DglcD1/D2
Mutants Overexpressing No-LOX or At-GOX2.
(A) and (B) Growth of the Synechocystis wild type (WT), single, and
double glcD mutants and the GOX-complemented strains DglcD1/D2-
No-LOX and DglcD1/D2-At-GOX2, respectively, was analyzed in liquid
media (A) and on agar plates (B) under ambient air (LC) conditions.
(C) Quantification of intracellular glycolate in cells of Synechocystis wild
type, double mutant DglcD1/D2, and the two complemented strains after
24 h at LC conditions. r.u., relative units.
Bar graphs represent mean and SD of three independent biological
replicates for each experiment. Statistically significant differences in the
growth rate compared with the wild type (asterisk) and corresponding
single mutant (double asterisk) cells are indicated (P < 0.05).
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DISCUSSION
GOX catalyzes the oxygen-dependent oxidation of glycolate to
glyoxylate and is known to be an essential peroxisomal enzyme
in photorespiratory metabolism in land plants (for example,
Clagett et al., 1949; Zelitch et al., 2009). Until recently, glycolate
oxidation in cyanobacteria and green algae was more or less ex-
clusively attributed to glycolate dehydrogenases (EC 1.1.99.14)
(Codd et al., 1969; Nelson and Tolbert, 1970; Frederick et al.,
1973; Beezley et al., 1976; Eisenhut et al., 2008a; Atteia et al.,
2009). These glycolate dehydrogenases are similar to the
GlcD subunit ofE. coli glycolate dehydrogenase, also sometimes
referred to as GOX (Pellicer et al., 1996), which allows for this
heterotrophic bacterium to use glycolate as a carbon source via
the glycerate pathway. In most cyanobacteria, this pathway
provides one of three routes for the recovery of photorespiratory
glycolate (Eisenhut et al., 2008a; Bauwe et al., 2010). By contrast,
glycolate oxidation via GOX was thought to be specific to land
plants and not present in other oxygenic phototrophs, with the
possible exception of some streptophyte algae (Stabenau and
Winkler, 2005; Chauvin et al., 2008). Therefore, the presence of
glycolate dehydrogenases was considered to be an ancestral
feature that was replaced by GOX in the land plant lineage. Our
phylogenetic and subsequent biochemical analyses provide
evidence that functional 2-hydroxy-acid oxidases with low
GOX activity are also present in green algae and in N2-fixing
cyanobacteria but that the Nostoc and Chlamydomonas en-
zymes No-LOX and Cr-LOX, respectively, use L-lactate rather
than glycolate as their preferred substrate. A close relationship
between GOX and LOX was not surprising because both en-
zymes use FMN as cofactors and short-chain hydroxyl acids as
substrates (Lindqvist and Brändén, 1989; Maeda-Yorita et al.,
1995; Leiros et al., 2006). Moreover, the phylogenetic relation of
cyanobacterial, algal, and plant GOX-like proteins implies that
the ancestor of the plant GOX family was acquired from the
primary endosymbiotic event. Therefore, the GOX family repre-
sents another example of the endosymbiotic transfer of proteins
for oxygenic photosynthesis and for the ability to detoxify photo-
respiratory glycolate, two closely related metabolic processes.
Our studies show that glycolate became the preferred sub-
strate at some point in the evolution of plants. The Vmax of No-
LOX for glycolate was;700-fold lower when compared with theplant GOX (Table 2). However, the affinity of No-LOX for glycolate
is similar to the reported values for GOX from pea (Pisum
Figure 5. Phenotypic Characterization of Nostoc Wild Type and Mutant Dlox Grown at Different Conditions.
(A) and (B) Cells were transferred from nitrate-containing medium BG11 (+N) into nitrate-free medium BG110 (-N) at time point 0 in liquid cultures under
CO2-enriched (HC) conditions (A) or onto agar plates at ambient air (LC) conditions (B). WT, wild type.
(C) Alternatively, cells in liquid cultures were transferred from nitrate-containing medium BG11 into nitrate-free medium BG110 supplemented with 10
mM HCO3� at time point 0 at microaerobic conditions obtained by bubbling with pure N2.Bar graphs represent mean and SD of five independent biological replicates for each experiment. Statistically significant differences between the wild
type and Dlox are indicated (*P < 0.05 and **P < 0.01).
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sativum), spinach, orMesostigma viride (Kerr and Groves, 1975;
Macheroux et al., 1992; Iwamoto and Ikawa, 2000) and sig-
nificantly higher than that of At-GOX2. These enzyme kinetics
data support the notion that the cyanobacterial GOX-like proteins
emerged from ancestral LOX proteins. In the anoxic Precambrian
ocean, fermentationwas the dominantmetabolicmode (Barnabas
et al., 1982), and these LOX proteins were likely involved in the
utilization of the available fermentation product L-lactate as ob-
served in extant anaerobic microbial mats (Anderson et al., 1987).
Apparently, during the evolution of plant GOX, the efficiency
of glycolate oxidationwas greatly improved, while the efficiency of
L-lactate oxidation was strongly decreased.
To mimic some of the alterations that were responsible for the
change of L-lactate to glycolate preference, we performed a
mutational analysis of No-LOX, using published information on
the structure of the active sites of the GOX and LOX enzymes
from other organisms (for example, Stenberg and Lindqvist,
1997). To this end, the amino acids at positions 82, 112, and 212
from No-LOX were exchanged for the corresponding amino
acids in plant GOX. Activity measurements with glycolate and
L-lactate as substrates revealed that all three positions affect
the relative substrate preference of No-LOX. The individual or
combined exchanges at positions 82 and 212 all improved the
reactivities of the respective No-LOX variants with glycolate
and, with the exception of the M82T mutation, decreased their
reactivities with L-lactate. The combination of these two muta-
tions in a double mutant resulted in a 10-fold higher GOX activity
and a 15-fold lower LOX/GOX ratio. These results suggest that
the No-LOX active site is optimized for the use of L-lactate as a
substrate. The F212V variant shows that the exchange of one
amino acid is capable of inducing threefold higher activity levels
with glycolate and 10-fold lower activity levels with L-lactate,
representing a threefold enhanced performance, compared with
Cr-LOX. In the triple-substituted No-LOX variant, the LOX/GOX
activity ratio was close to that of the plant enzyme. However, the
maximum GOX activity (0.6 6 0.1 mmol min21 mg21 in L112W/F212V) achieved by these alterations inNo-LOXwas still 40 times
lower than that of At-GOX2, indicating that a number of additional
mutations and corresponding fine adjustments must have con-
tributed to the evolution of specific GOX enzymes in land plants.
Compared with land plants, the overall photorespiratory pro-
duction of glycolate is much lower in cyanobacteria and green
algae due to the presence of CCMs in these organisms (Giordano
et al., 2005; Huege et al., 2011). It appears that the low glycolate
synthesis rate in cyanobacteria and the relatively high affinity of
No-LOX for glycolate (eightfold lower Km in comparison with At-
GOX2) was essential for the successful complementation of the
HCR phenotype of the DglcD1/D2 Synechocystis double mutant.
However, the very low GOX activities of the proteins from Nostoc
and Chlamydomonas indicate that these enzymes are unlikely to
be involved in photorespiratory metabolism. Instead, our analysis
of the Nostoc Dlox mutant supports earlier studies, suggesting
that GlcDs (in Nostoc encoded by alr5269 - glcD1 and all4443 -
glcD2) are responsible for the photorespiratory glycolate-to-
glyoxylate conversion in cyanobacteria and green algae (Marek
and Spalding, 1991; Nakamura et al., 2005; Eisenhut et al., 2008a).
The Chlamydomonas GlcD seems to be located in the mitochon-
drion and not in the peroxisomes, which is where the GOX of land
plants is confined (Atteia et al., 2009) and probably where Cr-LOX
is also confined (Shinozaki et al., 2009). Interestingly, a GlcD-like
enzyme is present in themitochondriaof land plants too (Bari et al.,
2004); however, this enzymeshows only low activitywith glycolate
and prefers D-lactate, indicating that it is unlikely to contribute to
photorespiration (Engqvist et al., 2009).
As mentioned above, our BLASTP searches with the translated
genomes of cyanobacteria revealed that GOX-like proteins are
exclusively present in cyanobacteria capable of N2 fixation. These
species included heterocystous (e.g., Nostoc), filamentous non-
heterocystous (e.g., Trichodesmium sp), and unicellular (e.g.,
Cyanothece spp) cyanobacteria that represent separate clades
within the cyanobacterial radiation (Gupta and Mathews, 2010).
Available data indicate that all ancient cyanobacteria were capa-
ble of fixing N2 until O2 accumulated in the environment ;2.5billion years ago (HartmannandBarnum,2010). As aconsequence
of O2 accumulation, and the almost simultaneous increase in
nitrate levels,manycyanobacteriamayhave lost the ability togrow
diazotrophically because they were unable to maintain nitroge-
nase activity in the presence of O2 (Falkowski, 1997; Berman-
Frank et al., 2003). Since the inactivation of nitrogenase by O2represents the main obstacle for N2 fixation, cyanobacteria have
evolved several elaborate O2 protection strategies, including
mechanisms to scavenge any environmental O2 that diffuses
with N2 (Fay, 1992; Berman-Frank et al., 2003; Walsby, 2007).
Respiration is one of these nitrogenase-protectingmechanisms. It
was shown that mutants of Nostoc defective in terminal oxidases
were not only affected in respiration but also lost their capability for
diazotrophic growth (Valladares et al., 2003, 2007). In addition,
respiration is necessary to provide the N2 fixation processes with
ATP and reductants in the absence of light (Fay, 1992). On the
other hand, sugar catabolism in the anoxic heterocyst could well
include anaerobic glycolysis to L-lactate, which is converted back
to pyruvate by No-LOX. Because this process consumes O2, it
could assist in protecting nitrogenase.
The absence of No-LOX did not impair the growth of the
corresponding Nostoc mutant, Dlox, in normal photorespiratory
Table 4. Nitrogenase Activity of Cells of the Nostoc Wild Type and
Mutant Dlox
Oxygen (%)
Nitrogenase Activity (mmol C2H4 g�1 Chla h�1)
24 h BG110 72 h BG110
Wild Type Dlox Wild Type Dlox
0 23.2 6 1.2 85.1 6 9.9* 55.9 6 10.6 76.1 6 42.7
5 24.1 6 5.5 35.0 6 2.0 52.9 6 44.7 68.8 6 18.6
10 34.2 6 23.9 41.5 6 1.2 51.2 6 3.0 29.9 6 10.5
15 20.4 6 9.5 19.1 6 20.8 44.3 6 9.4 20.3 6 14.3
20 141.8 6 29.1 44.8 6 1.8 86.9 6 0.4 47.1 6 3.7*
30 29.8 6 13.9 19.7 6 10.7 50.6 6 17.9 23.3 6 18.7
Nitrogenase activity of intact filaments was measured with cell suspen-
sions incubated for 24 and 72 h in nitrate-free medium BG110. The
filaments were incubated with different O2 (0 to 30%) and N2 (80 to 50%)
concentrations in the presence of 20% acetylene for 2 h. Data represent
mean and SD of at least three individual experiments. Statistically signif-
icant differences from the wild type are indicated by asterisks (P < 0.05).
Chla, chlorophyll a.
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conditions with nitrate-containing medium. However, the mutant
was unable to grow diazotrophically, indicating that No-LOX
contributes to N2 fixation in Nostoc, possibly by scavenging O2from the heterocysts. This suggestion is based on two central
experiments summarized in Figure 5 and Table 4. First, in contrast
with wild-type Nostoc, the mutant Dlox is unable to grow without
combined N sources in air but grows under microaerobic condi-
tions. Second, nitrogenase activity of the Dlox reached maximal
levels under microaerobic conditions and was inhibited by in-
creasing O2 concentrations, whereas the wild type showed
optimum nitrogenase activity at 20% O2 (Table 4). The lower
nitrogenase activities detected at lowO2 concentrations inNostoc
wild type is probably related to the essential role of respiration in
the supply of energy to nitrogenase. The unicellularCrocosphaera
watsonii also showed maximal N2 fixation in the presence of
oxygen (5%O2), whereas anoxic conditions completely abolished
nitrogenase activity (Compaoré and Stal, 2010). Although not fully
identical, the oxygen sensitivity of the Dloxmutant is similar to that
of a Nostoc mutant in which the two N-regulated terminal respi-
ratory oxidases were deleted, resulting in a complete loss of
nitrogenase activity (Valladares et al., 2003, 2007). Interestingly,
the affinity of No-LOX for the cosubstrate O2 was in the same
range as cyanobacterial respiratory cytochrome oxidases (Jensen
and Cox, 1983; Pils and Schmetterer, 2001) and would be suffi-
cient to decrease the O2 content inside heterocysts below levels
toxic for nitrogenase. However, in Dlox, nitrogenase was only
partially inactivated under comparable conditions, even after the
incubation of the filaments in air for 72 h.
In addition to the O2-scavenging function, No-LOX could also
be important for the redirection of lactate originating from an-
aerobic glycolysis into gluconeogenesis, similar to the well-
known Cori cycle of mammals. The energy requirements of this
process would then suggest the cooperation of the heterocysts
with the photosynthetic cells of Nostoc. In both cases, the
generated H2O2 could be removed by rubredoxin-like proteins,
such as RbrA. These proteins use H2 as a reducing agent, which
is produced as a byproduct of N2 fixation (Zhao et al., 2007). Our
current hypothesis, that No-LOX represents one of several
oxygen-scavenging mechanisms in Nostoc, needs additional
support by more data.
In summary, it appears that cyanobacteria that maintained
their capacity for N2 fixation also kept the oxygen-consuming
enzyme LOX as one of the mechanisms to protect nitrogenase
from O2 inactivation. Conversely, the loss of nitrogenase was
accompanied by the loss of lox genes. Whether this indicates
that the primary endosymbiont was still able to fix N2, a capability
which was later lost, cannot be determined from our data. Our
data also suggest that the enzyme acquired its specificity for
glycolate and became the photorespiratory GOX sometime after
the last common ancestor of Chlamydomonas and land plants.
METHODS
Strains and Culture Conditions
The Glc-tolerant wild-type strain Synechocystis sp PCC 6803 was
obtained from N. Murata (National Institute for Basic Biology, Okazaki,
Japan).Nostoc (Anabaena) sp PCC 7120 wild type was obtained from the
Pasteur Culture Collection (Paris). The Chlamydomonas reinhardtii strain
137c mt+ was obtained from Y. Tsubo (Kobe University, Hyogo, Japan)
via H. Takeda (Niigata University, Niigata, Japan).
The construction of the Synechocystis single mutants DglcD1
(sll0404::Km) and DglcD2 (slr0806::Sp) and the double mutant DglcD1/
D2 was previously described (Eisenhut et al., 2006, 2008a). Axenic
cultures were grown on Petri dishes at 308Cunder constant illumination of
30 mmol photons m22 s21 (warm white light; Osram L58 W32/3) using
agar-solidified BG11medium (Rippka et al., 1979) buffered to pH 8.0 with
20 mM TES-KOH. Transformants were initially selected on media con-
taining one of the following: 10mg L21 kanamycin (Km), 4 mg L21 Sp, or 5
mg L21 chloramphenicol (Cm). The segregation of clones and cultivation
of mutants were done with one of the following: 50mg L21 Km, 20mg L21
Sp, or 5 mg L21 Cm. All strains are listed in the Supplemental Table
1 online.
For the physiological characterization of Synechocystis mutants,
axenic cultures of a defined optical density at 750 nm (OD750) of 0.8
were grown photoautotrophically in batch cultures at 298C under contin-
uous illumination of 100 mmol photons m22 s21 (warm white light; Osram
L58 W32/3) and bubbled with air enriched with CO2 (5%, defined as high
inorganic carbon [HC]) in BG11 medium at pH 8.0 or bubbled with air
(0.035% CO2, defined as low inorganic carbon [LC]) in BG11 medium at
pH 7.0.
For the physiological characterization of Nostoc mutants, axenic cul-
tures of an OD750 1.2 were grown photoautotrophically in batch cultures
at 298C under continuous illumination of 40 mmol photons m22 s21 at HC
in standard BG11 medium containing nitrate as combined N source or in
nitrate-free BG11 medium (BG110) at pH 8.0. To grow Nostoc under
microaerobic conditions, the cells were bubbled with N2 in BG110supplemented with 10 mM NaHCO3.
For the cloning of the lox cDNA from C. reinhardtii, the wild-type strain
(137c mt+) was grown photoautotrophically with 500 mL of HS medium
(Sueoka, 1960) under continuous illumination of 200 mmol photons m22
s21 at 258C. The culture was bubbled with ambient air (LC) at a rate of 200
mL min21.
Growth wasmonitored bymeasurements of the OD750. Photosynthetic
pigment concentrations were estimated as described by Huckauf et al.
(2000). Contamination by heterotrophic bacteria was checked by spread-
ing of 0.2 mL of culture on Luria-Bertani plates.
DNAManipulation and Generation of Protein Expression
Strains or Mutants
Total DNA from Synechocystis and Nostoc was isolated according
to Hagemann et al. (1997). All other DNA techniques, such as plasmid
isolation, transformation of Escherichia coli, ligations, and restriction
analysis (restriction enzymes were obtained from Fermentas or New
England Biolabs), were standard methods.
To generate overexpressing strains of E. coli as well as of Synecho-
cystis, we amplified the coding sequences of the selected genes by PCR
using DNAofNostoc or cDNA ofArabidopsis thaliana as templates, gene-
specific primers with added appropriate cleavage sites (see Supplemen-
tal Table 2 online), and proofreading Elongase enzyme (Invitrogen).
For the cloning of the C. reinhardtii lox cDNA, mRNA was isolated from
wild-type cells in exponential growth phase using RNAgents Total RNA
isolation system (Promega) and PolyATract mRNA isolation system
(Promega). One microgram of mRNA was reverse transcribed using
PowerScript reverse transcriptase (Clontech) with a gene-specific primer
(CrLOX_R1; see Supplemental Table 2 online). The resulting cDNA
solution was diluted (1:10) with TE buffer and then used as template for
PCR amplification. A cDNA fragment containing LOX coding region was
amplified using PrimeSTAR GXL DNA polymerase (TaKaRa BIO) with a
primer pair of CrLOX_F1 and CrLOX_R2 (see Supplemental Table 2
online). The amplified fragment was then cloned into the pGEMT-easy
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vector (Promega) and sequenced with a BigDye Terminator version 3.1
cycle sequence kit (Applied Biosystems).
Site-specificmutants of No-LOX (All0170) were generated by PCRwith
Phusion high-fidelity polymerase using phosphorylated mutagenesis
primers (see Supplemental Table 2 online) and the cloned wild-type
all0170 gene as template. First, single amino acid exchanges at the
positions 82, 112, or 212 of No-LOX were introduced. Second, double
amino acid exchanges were made using the single-mutant constructs as
template, and finally the triple amino acid exchange was obtained using
the double mutant construct (M82T/L112W). The site-specific mutagen-
esis constructs were self-ligated and transformed into E. coli. All con-
structs were verified by restriction and sequence analyses.
For expression of the genes from Nostoc (No-LOX, lox, all0170), Arab-
idopsis (At-GOX2, At3g14415), C. reinhardtii (Cr-LOX, XP_001703481;
AB610509), and the site-specificmutants of No-LOX inE. coli, the verified
coding sequences were transferred into the expression vector pET-28a
(Novagen) using NdeI/EcoRI for No-LOX, Cr-LOX, and the site-specific
No-LOX mutants and BamHI/SalI for At-GOX2, respectively (see Sup-
plemental Table 2 online). The verified recombinant pET-28a vectors were
transformed into E. coli strain BL21 DE3.
For expression of No-LOX and At-GOX2 in the DglcD1/D2 double
mutant of Synechocystis, the coding sequences were transferred into the
expression vector pAII downstreamof the psba2 promoter (Lagarde et al.,
2000) using NdeI/KpnI (see Supplemental Table 2 online). For clone
selection, the Cm resistance cartridge from pCAT was cloned into pAII
downstream of the psba2 promoter and the inserted genes using the
BamHI cleavage site. The generated plasmids were transformed into the
HC-requiring DglcD1/D2 double mutant of Synechocystis (Eisenhut et al.,
2008a). Transformants were selected on agar plates supplemented with
Cm at LC conditions.
To generate the Nostocmutant Dlox (Dall0170::Sp), a 975-bp fragment
of the upstream region and a 898-bp fragment of the downstream region
of the gene all0170 were amplified using specific primers with cleavage
sites for XhoI/BamHI and BamHI/SacI, respectively (see Supplemental
Table 2 online). The upstream region was cloned into pDrive (Qiagen)
using XhoI and ApaI generating pDrive-all0170-up. Subsequently, the
downstream region was cloned into pDrive-all0170-up after cleavage
with ApaI and SacI. The adjacent regions of all0170 were cloned into the
cargo plasmid pRL271 (kindly provided by W. Lockau, Humboldt Uni-
versity Berlin, Germany) using XhoI and SacI. The cargo plasmid pRL271
is a cloning vector for sacB-mediated positive selection of targeted gene
replacement and double recombinants in cyanobacteria (Black et al.,
1993). Finally, the Sp resistance cartridge from pAII::Sp was cloned
between the all0170 up- and downstream regions to generate pRL271-
all0170::Sp using BamHI cleavage sites (see Supplemental Figure 3
online). The verified construct was conjugated into Nostoc as previously
described (Cai and Wolk, 1990). Double recombinant filaments were
selected on agar plates supplemented with Sp and/or Suc.
Purification of Recombinant GOX Proteins after
Overexpression in E. coli
E. coli BL21 strains containing pET-28a-NoLOX or site-specific No-LOX
mutagenesis constructs, pET-28a-CrLOX, and pET28a-AtGOX2 were
grown in Luria-Bertani medium to an OD750 of 0.6. Gene expression was
induced by addition of 1 mM isopropyl b-1-D-thiogalactopyranoside for
16 h at 308C. The fusion proteins were purified via their N-terminal His-
tags using Ni-NTA Sepharose according to the protocol of the supplier
(Invitrogen). The cells were harvested and resuspended in 20 mM Tris-
HCl, pH 8.0, containing 500 mM NaCl, 1 mM DTT, and 0.1 mM FMN.
Protein was extracted by ultrasonic treatments (2 3 60 s, 90 W) in ice.
Soluble protein extracts were used for affinity chromatography onNi-NTA
Sepharose using 20mMTris-HCl, pH 8.0, containing 500mMNaCl, 1mM
DTT, and 0.1 mM FMN supplemented with 40 up to 80 mM imidazole as
washing buffers. The same buffer supplemented with 200 mM imidazole
was used as elution buffer. The eluted proteins were combined and
desalted using PD-10 columns (GE Healthcare). Finally, the recombinant
enzymes were dissolved in 20 mM Tris-HCl, pH 8.0, with 1 mM DTT and
0.1 mM FMN. The eluted proteins were checked regarding purity using
SDS-PAGE and staining by Coomassie Brilliant Blue (see Supplemental
Figure 2 online) and subsequently used for enzyme measurements and
antibody production.
EnzymeMeasurements
Enzyme activity was measured in 100 mM Tris-HCl, pH 8.0, containing
5 mM MgCl2, 0.5 mM EDTA, 1 mM DTT, 0.1 mM FMN, and 0.0083%
Triton-X 100 at 308C. The O2 consumption of LOX and GOX was assayed
using Hansatech oxygen electrodes (Oxygraph). The assay was started
by adding of different substrates after 3 min of equilibration. One unit of
enzyme activity was defined by the formation of 1 mmol of O2 in 1 min at
308C. The affinity of No-LOX for oxygen was estimated according to the
methods of Macheroux et al. (1993) and Mills et al. (2001) using different
L-lactate concentrations (1 to 10 mM) and different enzyme amounts.
Additionally, hydrogen peroxide production was measured using 3,39,5,59-
tetramethylbenzidine according to Josephy et al. (1982) and Kireyko et al.
(2006). The recombinant proteins were incubated for 5 min without
(control) or 1 mM substrate in the enzyme buffer described above.
H2O2 was quantified by adding 2 mM 3,39,5,59-tetramethylbenzidine and
2 units of horseradish peroxidase. Kinetic parameters were calculated by
nonlinear regression fit to the Michaelis-Menten equation (Sigma Plot
software) or by linear regression analysis of the double-reciprocal data
pairs (Lineweaver-Burk).
Immunoblotting and Antibody Purification
Polyclonal antibodies specific for No-LOX or At-GOX2 were raised in
rabbits using the corresponding pure recombinant proteins as antigens
(Seqlab). To increase the specificity of the antibodies, they were purified
as follows. About 1 mg of recombinant protein was separated on 12%
acrylamide gels and blotted onto nylonmembranes (GEHealthcare). After
visualization by Ponceau S staining (0.2% Ponceau S in 0.25% acetic
acid), the respective bands were cut and the membrane pieces were
washed in glycine buffer (100 mM glycine-HCl, pH 2.5) for 5 min. After
washing, the membranes were blocked with 3% BSA for 60 min and
incubated with the antisera after threefold dilution in PBS overnight at 48C
while shaking. After washing with PBS, the antibodies were eluted with
1 mL glycine buffer for 10 min. The elution was repeated and the fractions
were pooled. To adjust the pH at ;7.0, 200 mL of 1 M Tris-HCl, pH 8.0,was added to the purified antibody solutions, and BSA (1 mg mL21) was
added as stabilizer.
For immunoblotting, 20mL of a culture of Synechocystis grown for 24 h
under HCor LCconditionswas harvested by centrifugation (3140 rcf, 48C,
10min). The cell pellet was immediately frozen and stored at2808C. Total
proteins were extracted by sonication in ice-cold 10 mM HEPES-NaOH,
pH 7.5, containing phenylmethylsulfonyl fluoride. Equal amounts of
soluble protein (10 mg) were separated by PAGE (12% polyacrylamide)
and subsequently blotted onto polyvinylidene fluoride membranes (Bio-
Rad). The cross-reacting bands were detected by the purified No-LOX-
or At-GOX2-antibodies (diluted 400- or 1000-fold) using horseradish
peroxidase–conjugated anti-rabbit IgG (Bio-Rad) as secondary antibody.
Quantification of Glycolate
Glycolate was determined using a metabolite targeted and quantitatively
standardized extended variant of the previously established gas chro-
matography–electron impact–time of flight–mass spectrometry pro-
filing analysis. Cells grown in liquid media at HC or LC conditions were
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harvested by fast filtration in the light and were immediately frozen in
liquid N2. The whole procedure has been described in detail previously
(Eisenhut et al., 2008b; Huege et al., 2011), but instead of MSTFA as
derivatization agent, TBDMS was used.
Nitrogenase Activity Measurements
Cultures of Nostoc wild type or Dlox mutant were shaken in 300-mL
Erlenmeyer flasks at 298C under continuous illumination (40 mmol pho-
tons m22 s21) in BG110 at pH 8.0 in air (LC). Two milliliters of culture were
placed in a 10-mL vial sealed with a rubber stopper and bubbled for 5min
with different ratios of O2 and N2 to achieve defined O2 concentrations in
the presence of 20% (v/v) acetylene using a gas-mixing system of mass
flow controllers (Brooks 5850S) and control unit (Brooks 0152) as de-
scribed earlier (Staal et al., 2001). After 2 h of incubation under the growth
conditions, ethylenewasmeasured in the head space of the vial using gas
chromatography (Chrompack CP9001) equipped with a flame ionization
detector (FID) and a wide-bore silica-fused (0.53-mm internal diameter)
Porapak U column (Chrompack). The carrier gas was N2 at 10 mL min21,
and the flows of H2 and air for the flame ionization detector were 30 and
300 mL min21, respectively. The temperatures for injector, detector, and
oven were 90, 120, and 558C, respectively.
All experiments (growth rates of different strains, enzyme activity mea-
surements, and metabolite contents) were repeated at least three time
using independent biological replicates. Statistically significant differences
estimated using Student’s t test.
Phylogenetic Analysis of GOXs and GOX-Like Proteins
To analyze the evolutionary history of the GOX enzymes in the plant 2PG
metabolism, proteins similar to the Arabidopsis GOX2 (At3g14415;
extracted from The Arabidopsis Information Resource database: http://
www.Arabidopsis.org/) were searched by the BLASTP (Altschul and
Lipman, 1990) algorithm. Among cyanobacteria, the gene all0170 from
Nostoc was identified to encode for a GOX-like protein (CyanoBase:
http://genome.kazusa.or.jp/cyanobase/). Subsequently, this protein se-
quence was used for further searches. The sequences were retrieved
from the following databases: (1) GenBank (http://www.ncbi.nlm.nih.gov/
genbank/), (2) PlantGDB (http://www.plantgdb.org/OsGDB/), (3) Cyano-
Base (http://genome.kazusa.or.jp/cyanobase), (4) Cyanidioschyzon mer-
olae genome browser (http://merolae.biol.s.u-tokyo.ac.jp/; Matsuzaki
et al., 2004), and (5) The Arabidopsis Information Resource (http://
www.Arabidopsis.org/). For phylogenetic analyses, the homologous
amino acid sequences were aligned using the ClustalW algorithm
(Thompson et al., 1994) integrated in the BioEdit Sequence alignment
editor (Hall, 1999). The alignment is available as Supplemental Data Set
1 online. The neighbor-joining tree of putative and biochemically charac-
terized LOX or GOX proteins was inferred using MEGA version 4.0
(Tamura et al., 2007). The Jones-Taylor-Thornton substitution model
(Jones et al., 1992) was selected assuming a gamma correction for
heterogeneity across sites. Reliability for internal branch was assessed
using the bootstrapping method (1000 bootstrap replicates). Graphical
representation and edition of the phylogenetic tree were performed with
TreeDyn (v198.3) (Chevenet et al., 2006).
Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL/CyanBase/DDBJ databases under the following
accession numbers: Arabidopsis GOX2 (At3g14415), Arabidopsis HAOX1
(At3g14130), Zea mays (ACN28768), Vitis vinifera (CAN67413), Spinacia
oleracea (AAA34030), Physcomitrella patens (XP_001769086), C. reinhardtii
CC-503 (XP_001703481, AB610509), Volvox carteri (XP_002946783), Ecto-
carpus siliculosus (CBN74053), Cyanothece sp PCC 7822 (YP_003890366),
Salmonella enterica strainGA-MM04042433 (ZP_03218859),Acidaminococ-
cus sp D21 (ZP_039818), Lactococcus lactis Il1403 (AAK05350), Weissella
paramesenteroides ATCC33313 (ZP_04782696), Aerococcus viridans
IF012219 (BAA09172),Enterococus faeciumDO (ZP_05714515),Oryza sativa
(Os07g0152900), Anabaena variabilis ATCC 29413 (Ava_1430), Nostoc
punctiforme ATCC 29133 (Npun_R5717), Trichodesmium erythraeum
IMS101 (Tery_2398), Cyanothece sp ATCC 51,142 (cce_1717), and Cyani-
dioschyzon merolae 10D (CMQ436C).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Amino Acid Sequence Alignment of GOX
and LOX.
Supplemental Figure 2. Purification of Recombinant Proteins.
Supplemental Figure 3. Genotypic Characterization of the Nostoc
Mutant Dlox.
Supplemental Table 1. Strains Used in this Work.
Supplemental Table 2. Primers Used in this Work.
Supplemental Data Set 1. Text File of the Alignment Used for the
Phylogenetic Analysis Shown in Figure 1.
ACKNOWLEDGMENTS
The excellent technical assistance of Manja Henneberg, Kathrin Jahnke,
and Klaudia Michl is greatly acknowledged. The generous help of
Wolfgang Lockau and his group in establishing genetics with Nostoc
7120 is highly appreciated. The work was supported by a grant from the
Deutsche Forschungsgemeinschaft to M.H. and the Forschergruppe
FOR 1186-Promics.
AUTHOR CONTRIBUTIONS
M.H. designed research. C.H., R.K., J.H., Y.T., and L.J.S., performed
research. C.H., R.K., J.H., L.J.S., Y.T., J.K., Y.S., H.B., andM.H. analyzed
data. C.H., R.K., L.J.S., J.K., Y.S., H.B., and M.H. wrote the article.
Received June 8, 2011; revised July 15, 2011; accepted July 19, 2011;
published August 9, 2011.
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